Study On Multiplex PCR For Rapid Detection Of Three Food-borne Bacterial Pathogens

The food security is a major public health problem and attracts more and more attention in the world. Food poisoning caused by bacteria hold a greater proportion in a variety of food safety incidents and bacterial food poisoning is the main factor which causes the food poisoning. Salmonella, Listeria monocytogenes and Yersinia enterocolitica are three food-borne pathogenic bacteria which can cause deadly diseases in humans and livestock, and they may contaminate various food. At present, detection of food-borne pathogens mainly uses the traditional isolation, culture and biochemical identification method in our country. It is incapable of detecting pathogens quickly and sensitively because of the complicated operation, time consuming and low sensitivity. Multiplex PCR has a broad prospect of application in pathogenic bacteria testing field and the simultaneous detection of several pathogens with a multiplex PCR approach which has the same basic principle as conventional PCR would save cost and time.In this study, three pairs of specific primers were designed according to the fimY gene of Salmonella, the hlyA gene of Listeria monocytogenes and the ail gene of Yersinia enterocolitica, thus the multiplex PCR was established to simultaneous diagnose three kinds of food-borne pathogenic bacteria. According to the influence factors of multiplex PCR, the orthogonal experimental design, L9(33) and L16(44) table, were used to optimize multiple PCR amplification system of three factors (three primer proportion) at three levels and four factors (Taq DNA polymerase, Mg2+, dNTPs and primers) at four levels. Then annealing temperature and the concentration of the buffer were optimized. As a result, the multiplex PCR reaction system was determined to be 25μL: 3.8μL 10~×PCR buffer, 1.5μL Mg2+(50 mmol/L), 3.0μL dNTPs(each 2.5 mmol/L), 0.3μL Taq DNA polymerase (5 U/μL), each primer 1.0μL(10~μmol/L) of Salmonella, each primer 1.0μL(10~μmol/L) of Listeria monocytogenes, each primer 1.0μL(10~μmol/L) of Yersinia enterocolitica, template 0.5μL,ddH20 8.9μL. The cycle parameters:Pre-degenerated at 94℃for 5 min, degenerated at 94℃for 45 s, annealing at 59.8℃for 45 s, extention at 72℃for 45 s, run 30 cycles, the final extention at 72℃for 10~ min.It is showed that this method is of better specificity through the primer specific experiment and the reaction specific experiment. Multiplex PCR products were confirmed by DNA sequencing. The results of sequencing were compared with the sequence of the target gene at GenBank and three homologies were all above 99% and multiplex PCR amplified products were certified. The sensitivity and specificity of multiplex PCR were compared with that of the simplex PCR assay method which was regard as a control. Under the above optimum experimental condition, the sensitivity of the simultaneous detection of the three bacterial pathogens with multiplex PCR was 10~2 CFU/mL for Salmonella, 10~3 CFU/mL for Listeria monocytogenes and Yersinia enterocolitica. Extracting genomic DNA with kit extraction, the detection limit of artificially contaminated pork was 10~3 CFU/g for Salmonella, 10~4 CFU/g for Listeria monocytogenes and Yersinia enterocolitica. The sensitivity and detection limit of the multiplex PCR were below that of the simplex PCR. The enrichmented culture was incubated for 9 hours after artificially contaminating pork, and the detection limit of the multiplex PCR assay for meat was up to 10~0 CFU/g.Compared with the national standard method, the multiple PCR have high sensitivity, specificity and coincidence rate for detection of the actual samples of the three pathogenic bacteria. In the study, multiplex PCR assay had been established and the assay was simple, rapid, exact, high-sensitive, low-cost, high-throughput and with strong practical application value. It will play an important part in controlling pathogen transmission and preventing the occurrence of food poisoning in future.